Trihydroxy Polyunsaturated Eicosanoid

ABSTRACT

The invention features methods for the preparation of naturally occurring trihydroxy polyunsaturated eicosanoids and their structural analogs. The invention further provides new derivatives and analogs of trihydroxy polyunsaturated eicosanoids that can be prepared according to these methods. The invention also provides compositions and methods using trihydroxy polyunsaturated eicosanoid derivatives for the prevention, amelioration and treatment of a variety of diseases or conditions associated with inflammation or inflammatory response, autoimmune diseases, rheumatoid arthritis, cardiovascular diseases, or abnormal cell proliferation or cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/093,757, filed Mar. 29, 2005, which is a continuation-in-part of U.S.patent application Ser. No. 10/405,924, filed on Apr. 1, 2003, now U.S.Pat. No. 6,949,664. U.S. patent application Ser. No. 10/405,924 claimsbenefit of priority under 35 U.S.C. § 119(e) to and U.S. ProvisionalPatent Application Ser. No. 60/369,543, filed on Apr. 1, 2002. Thecontent of each of these applications are hereby incorporated in itsentirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

The U.S. Government may have certain rights in this invention pursuantto Grant No. PO1-DE13499 (Subcontract) awarded by the NationalInstitutes of Health.

FIELD OF THE INVENTION

This invention relates to trihydroxy polyunsaturated eicosanoidderivatives and methods for the preparation of such compounds and theirstructural analogs. This invention also relates to compounds,compositions and methods using trihydroxy polyunsaturated eicosanoidderivatives for the prevention, amelioration and treatment of a varietyof diseases or conditions associated with inflammation or inflammatoryresponse, autoimmune diseases, rheumatoid arthritis, cardiovasculardiseases, or abnormal cell proliferation or cancer.

BACKGROUND OF THE INVENTION

The present invention provides methods for preparing lipid mediatorsrelated to ω-3 polyunsaturated fatty acids (PUFA), which have potentialuse in the development of new pharmaceuticals based on thewell-established beneficial effects of PUFA.

It has long been suggested that dietary ω-3 polyunsaturated fatty acids(PUFA) (De Caterina, R., Endres, S.; Kristensen, S. D.; Schmidt, E. B.,(eds). ω-3 Fatty Acids and Vascular Disease, Springer-Verlag, London.166 pp. 1993; Gill, I., and Valivety, R. (1997), Trends in Biotechnology15, 401-409;) have beneficial effects in human health and in theprevention of various diseases, including inflammation and autoimmunediseases (Simopoulos, A. P. (2002), J. Am. Coll. Nutrition 21, 495-505),rheumatoid arthritis (Cleland, L. G., James, M. J., and Proudman, S. M.(2003), Drugs 63, 845-853), cardiovascular diseases (Billman, G. E., etal. Circulation. 1999, 99, 2452; Harper, C. R., and Jacobson, T. A.(2001) Arch. Intern. Med. 161, 2185-2192), and cancer (Iigo, M. et al,Br. J. Cancer, 1997, 75, 650; Larsson, S. C., Kumlin, M.,Ingelman-Sundberg, M., and Wolk, A. (2004), Am. J. Clin. Nutr. 79,935-945).

Eicosapentaenoic acid (C20:5), the major PUFA in fish oil, was shown toform prostaglandins (PG), leukotrienes (LT) and other eicosanoids thatare similar to those derived from arachidonic acid (C20:4). Thedifferent biological properties of these molecules were considered to beresponsible for the role of PUFA. Despite numerous studies in this area,however, the molecular mechanisms for the actions of PUFA remainunknown.

The conversion of arachidonic acid (C20:4) to a variety of bioactiveeicosanoids, including prostaglandins (PG), leukotrienes (LT) andlipoxins (LX) is well known (Nicolaou, K. C.; Ramphal, J. Y.; Petasis,N. A.; Serhan, C. N. Angew. Chem. Int. Ed. Engl. 1991, 30, 1100). It wasrecently demonstrated (Serhan, C. N. et al. J. Exp. Med. 2000. 192,1197) that human endothelial cells with up-regulated COX-2 treated withaspirin convert ω-3 polyunsaturated fatty acids to 18R-HEPE as well as15R-HEPE. While 15R-HEPE led to the 5-series lipoxins (15R-LXA₅),18R-HEPE led to 5S,12R,18R-triHEPE (1), a novel trihydroxy-eicosanoidrelated to the structure of LTB₄. Due to their role in the resolution ofinflammation, compounds of this type were named Resolvins (Serhan, C.N.; et al, J. Exp. Med. 2002, 196, 1025; Serhan, C. N. (2004) HistochemCell Biol (2004) 122:305-321), while compound 1 was named Resolvin E1.

The formation of these trihydroxy polyunsaturated eicosanoids from PUFAsuggests a novel mechanism for the therapeutic benefits of PUFA withmajor implications for new therapeutic approaches to a variety ofdiseases. Methods for the preparation of such compounds, therefore, areof great importance to the development of new therapeutic agents.Furthermore, the development of structural derivatives of thesecompounds may be useful for the optimization of their pharmacologicalprofile and other desirable drug-like properties.

SUMMARY

The invention features methods for the preparation of naturallyoccurring trihydroxy polyunsaturated eicosanoids and their structuralanalogs. The invention further provides new derivatives of trihydroxypolyunsaturated eicosanoids that can be prepared according to thesemethods.

In general, in one aspect, the invention features methods of preparingtrihydroxy polyunsaturated eicosanoids, such as 1, as outlined inScheme 1. The two (Z) C═C bonds can be formed via selectivehydrogenation of the bis-alkynyl precursor 2. Compound 2 can be preparedvia a palladium-mediated coupling (coupling step a) betweenintermediates 3 and 4, where X is Br, or I. Compound 4 can be preparedvia the olefination of aldehyde 5, which is readily available fromprotected epoxide 6. Intermediate 3 can be prepared in several differentways, as discussed below, from precursors 7 and 8, while compound 8 canreadily prepared from protected epoxide 9.

The invention also provides methods for the preparation of compounds ofthe general formula 3, which can be used to prepare trihydroxypolyunsaturated eicosanoids or their analogs. Compound 3 can be preparedin several different ways, as outlined in Scheme 2.

According to Method A, compound 3 can be prepared via the addition of anallenyl reagent 11 (M is magnesium, zinc, copper, tin, silicon or boron)to precursor 12, which is readily available via the Pd-coupling betweenthe known bromide 13 and the known alkyne 7.

According to Method B, compound 3 is prepared from precursor 10, whichis produced via Pd-mediated coupling (coupling process b) of 7 withintermediate 14. Compound 14, can be prepared via Pd-coupling (couplingprocess c) between 15 and precursor 16, which can be prepared via theolefination (coupling process d) of aldehyde intermediate 8.Alternatively, compound 14, can be prepared via a Wittig-type reaction(coupling process d) between 20 and aldehyde 8. Compound 14 can also beprepared via silylation of its alkyne precursor 21, which can be formedvia addition to the aldehyde 22 (coupling process a).

According to Method C, precursor 10, is formed via the Pd-coupling(coupling process c) between 16 and alkenyl boron compound 17, which isreadily available via the Pd-coupling (coupling process c) betweenalkenyl boron compound 18 and intermediate 7.

Finally, according to Method D, compound 10, is prepared via thealkenylation (coupling process d) of aldehyde intermediate 8 withphosphonate intermediate 19, which is readily available via thePd-coupling (coupling process b) between the compound 20 with 7.

In another aspect, the invention features methods for the synthesis ofcompounds having the general formulas 23 and 24, as outlined in Scheme3. Compound 23 can be prepared via the selective hydrogenation ofcompound 24, which can be produced via a Sonogashira-type coupling amongcompounds 25 and 26:

-   -   wherein:    -   R^(a), R^(b) and R^(c), are independently selected from the        group consisting of hydrogen, alkyl, aryl, heteroaryl, acyl,        silyl, alkoxyacyl and aminoacyl;    -   R¹, R² and R³ are independently selected from the group        consisting of hydrogen, alkyl, perfluoroalkyl, aryl and        heteroaryl;    -   Q is selected from the group consisting of:        -   —C(O)-A, —SO₂-A, —PO(OR)-A, where A is hydroxy, alkoxy,            aryloxy, amino, alkylamino, dialkylamino, or —OM, where M is            a cation selected from the group consisting of ammonium,            tetra-alkyl ammonium, Na, K, Mg, and Zn, and R is hydroxyl            or alkoxy;    -   Y, Z and W are linkers independently selected from the group        consisting of a ring containing up to 20 atoms and a chain of up        to 20 atoms, provided that Y, Z and W can independently include        one or more nitrogen, oxygen, sulfur or phosphorous atoms, and        further provided that Y, Z and W can independently include one        or more substituents selected from the group consisting of        hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro,        iodo, bromo, fluoro, hydroxy, alkoxy, aryloxy, carboxy, amino,        alkylamino, dialkylamino, acylamino, carboxamido, cyano, oxo,        thio, alkylthio, arylthio, acylthio, alkylsulfonate,        arylsulfonate, phosphoryl, and sulfonyl, and further provided        that Y, Z and W can also contain one or more fused carbocyclic,        heterocyclic, aryl or heteroaryl rings, and provided that all        linkers Y are connected to the adjacent C(R)OR group via a        carbon atom;    -   X is Cl, Br or I; and    -   G is selected from the group consisting of hydrogen, alkyl,        perfluoroalkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro,        iodo, bromo, fluoro, hydroxy, alkoxy, aryloxy, carboxy, amino,        alkylamino, dialkylamino, acylamino, and carboxamido

The invention also provides compounds and compositions containingsynthetic analogs of trihydroxy polyunsaturated eicosanoids that aresynthetic derivatives or analogs of compound 1 and exhibit improvedchemical and biological properties. The provided compounds includederivatives having the general formulas 23 and 24:

wherein,

-   -   A is hydroxy, alkoxy, aryloxy, amino, alkylamino, dialkylamino,        or —OM, where M is a cation selected from a group consisting of        ammonium, tetra-alkyl ammonium, Na, K, Mg, or Zn;    -   Ra, Rb and Rc, are independently selected from a group that        consists of hydrogen, alkyl, aryl, heteroaryl, acyl, silyl,        alkoxyacyl or aminoacyl;    -   R¹, R² and R³ are independently selected from a group that        consists of hydrogen, alkyl, perfluoroalkyl, aryl or heteroaryl;    -   Q is selected from a group that consists of:        -   —C(O)-A, —SO2-A, —PO(OR)-A, where A is hydroxy, alkoxy,            aryloxy, amino, alkylamino, dialkylamino, or —OM, where M is            a cation selected from a group consisting of ammonium,            tetra-alkyl ammonium, Na, K, Mg, or Zn; and R is hydroxyl or            alkoxy;    -   Y, Z and W are linkers selected from a group consisting of a        ring or a chain of up to 20 atoms that may include one or more        nitrogen, oxygen, sulfur or phosphorous atoms, provided that        linker A can have one or more substituents selected from the        group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, chloro, iodo, bromo, fluoro, hydroxy, alkoxy,        aryloxy, carboxy, amino, alkylamino, dialkylamino, acylamino,        carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio,        alkylsulfonate, arylsulfonate, phosphoryl, and sulfonyl, and        further provided that the linker may also contain one or more        fused rings, including carbocyclic, heterocyclic, aryl or        heteroaryl rings, provided that all linkers Y are connected to        the adjacent C(R)OR group via a carbon atom;    -   G is selected from a group that consists of hydrogen, alkyl,        perfluoroalkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro,        iodo, bromo, fluoro, hydroxy, alkoxy, aryloxy, carboxy, amino,        alkylamino, dialkylamino, acylamino, and carboxamido.

In other aspects, the invention also features pharmaceuticalcompositions including the compounds of the invention, as well astherapeutic uses for such compounds and compositions in treating and/orpreventing a disease or condition associated with inflammation orinflammatory response, autoimmune diseases, rheumatoid arthritis,cardiovascular diseases, or abnormal cell proliferation or cancer.

The details of one or more embodiments of the invention are set forth inthe description below. Unless otherwise defined, all technical andscientific terms used herein have the meaning commonly understood by oneof ordinary skill in the art to which this invention belongs. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. Other features and advantages of the invention will becomeapparent from the description and the claims.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used in this specification, alkyl groups can includestraight-chained, branched and cyclic alkyl radicals containing up toabout 20 carbons. Suitable alkyl groups may be saturated or unsaturated.Further, an alkyl may also be substituted one or more times on one ormore carbons with one or more substituents selected from the groupconsisting of C1-C6 alkyl, C3-C6 heterocycle, aryl, halo, hydroxy,amino, alkoxy and sulfonyl. Additionally, an alkyl group may contain upto 10 heteroatoms or heteroatom substituents. Suitable heteroatomsinclude nitrogen, oxygen, sulfur and phosphorous.

As used in this specification, aryl groups are aryl radicals which maycontain up to 10 heteroatoms. An aryl group may also be optionallysubstituted one or more times with an aryl group or a lower alkyl groupand it may be also fused to other aryl or cycloalkyl rings. Suitablearyl groups include, for example, phenyl, naphthyl, tolyl, imidazolyl,pyridyl, pyrroyl, thienyl, pyrimidyl, thiazolyl and furyl groups.

As used in this specification, a ring is defined as having up to 20atoms that may include one or more nitrogen, oxygen, sulfur orphosphorous atoms, provided that the ring can have one or moresubstituents selected from the group consisting of hydrogen, alkyl,allyl, alkenyl, alkynyl, aryl, heteroaryl, chloro, iodo, bromo, fluoro,hydroxy, alkoxy, aryloxy, carboxy, amino, alkylamino, dialkylamino,acylamino, carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio,alkylsulfonate, arylsulfonate, phosphoryl, and sulfonyl, and furtherprovided that the ring may also contain one or more fused rings,including carbocyclic, heterocyclic, aryl or heteroaryl rings.

As used herein, “alkylene” refers to a straight, branched or cyclic, incertain embodiments straight or branched, divalent aliphatic hydrocarbongroup, in one embodiment having from 1 to about 20 carbon atoms, inanother embodiment having from 1 to 12 carbons. In a further embodimentalkylene includes lower alkylene. There may be optionally inserted alongthe alkylene group one or more oxygen, sulphur or substituted orunsubstituted nitrogen atoms, where the nitrogen substituent is alkyl.Alkylene groups include, but are not limited to, methylene (—CH₂—),ethylene (—CH₂CH₂—), propylene (—(CH₂)₃—), methylenedioxy (—O—CH₂—O—)and ethylenedioxy (—O—(CH₂)₂—O—). The term “lower alkylene” refers toalkylene groups having 1 to 6 carbons. In certain embodiments, alkylenegroups are lower alkylene, including alkylene of 1 to 3 carbon atoms.

Methods for Preparing Trihydroxy Polyunsaturated Eicosanoids and Analogs

In general, in one aspect, the invention features methods of preparingtrihydroxy polyunsaturated eicosanoids, such as 1, as outlined inScheme 1. This strategy is highly convergent and the two (Z) C═C bondscan be generated at the last step and thereby enhancing the stabilityand stereochemical integrity of the product. The two (Z) C═C bonds canbe formed via selective hydrogenation of the bis-alkynyl precursor 2.The selective hydrogenation can be performed using hydrogen and Lindlarcatalyst, or by using activated zinc in the presence of an alcohol suchas methanol, or using an aqueous medium. The activated zinc reagentsuitable for this process can be prepared from zinc, a copper salt, suchas copper acetate and a silver salt, such as silver nitrate according toliterature procedures (Boland, W. et al (1987) Helv. Chim. Acta 1987,70, 1025; Alami, M. et al. (1997) Tetrahedron Asym., 8, 2949; Rodriguez,A. R. et al (2001) Tetrahedron Lett., 42, 6057).

Compound 2 can be prepared via a palladium-mediated coupling (couplingstep a) between intermediates 3 and 4, where X is Br, or I. Compound 4can be prepared via the olefination of aldehyde 4, which is readilyavailable from protected epoxide 6. Intermediate 3 can be prepared inseveral different ways, as discussed below, from precursors 7 and 8,while compound 8 can readily prepared from protected epoxide 9.

The invention also provides methods for the preparation of compounds ofthe general formula 3, which can be used to prepare trihydroxypolyunsaturated eicosanoids or their analogs. Compound 3 can be preparedin several different ways, as outlined in Scheme 2.

According to Method A, compound 3 can be prepared via the addition of anallenyl reagent 11 (M is magnesium, zinc, copper, tin, silicon or boron)to precursor 12, which is readily available via the Pd-coupling betweenthe known bromide 13 and the known alkyne 7.

According to Method B, compound 3 is prepared from precursor 10, whichis produced via Pd-mediated coupling (coupling process b) of 7 withintermediate 14. Compound 14, can be prepared via Pd-coupling (couplingprocess c) between 15 and precursor 16, which can be prepared via theolefination (coupling process d) of aldehyde intermediate 8.Alternatively, compound 14, can be prepared via a Wittig-type reaction(coupling process d) between 20 and aldehyde 8. Compound 14 can also beprepared via silylation of its alkyne precursor 21, which can be formedvia addition to the aldehyde 22 (coupling process a).

According to Method C, precursor 10, is formed via the Pd-coupling(coupling process c) between 16 and alkenyl boron compound 17, which isreadily available via the Pd-coupling (coupling process c) betweenalkenyl boron compound 18 and intermediate 7.

Finally, according to Method D, compound 10, is prepared via thealkenylation (coupling process d) of aldehyde intermediate 8 withphosphonate intermediate 19, which is readily available via thePd-coupling (coupling process b) between the compound 20 with 7.

The present invention involves several distinct building blocks whichcan be readily prepared as described below.

Scheme 4 shows the synthesis of building blocks of type 4, while Scheme5 shows the synthesis of building blocks of type 8 and 16. In both casesthe stereochemistry of these building blocks is establishedunambiguously from the starting glycidol and it is retained throughoutthe synthesis, allowing the synthesis of products with highstereochemical purity.

Scheme 6 shows a method for the synthesis of intermediate of type 7 withhigh stereochemical purity.

The combination of these building blocks to form key intermediate 3, canbe done in a variety of ways. Scheme 7 shows a strategy according toMethod A (Scheme 2), whereby the alkyne intermediate of type 7, can becoupled with a dienyl bromide-alcohol to give a product that can beoxidized to an aldehyde. Addition of allenyl boronic acid derivative,according to chemistry reported by Yamamoto (Ikeda, N.; Arai, I.;Yamamoto, H. J. Am. Chem. Soc. 1986, 108, 483.) forms the intermediateof type 3, in good overall yield, but with modest stereocontrol.

Scheme 8 shows an alternative way to prepare the intermediate of type 3is via an intermediate of type 10. According to Method B (Scheme 2)Negishi-type coupling of intermediate of type 16 followed by Sonogashiracoupling with intermediate of type 7 gives the intermediate of type 10,which can be de-silylated to form the key intermediate of type 3.

Another approach according to Method C for the preparation of 10, isshown in Scheme 9. Sonogashira coupling, followed by a Suzuki couplinggives the final product. This iterative coupling can be done in asequential manner and it is possible to do this in one pot.

Scheme 10 shows one of the most effective ways to make intermediates oftype 10, which can be produced via Pd-mediated coupling of 7 withintermediate 14. Compound 14 can be prepared via a Wittig-type reactionbetween phosphonate 20 and aldehyde 8, followed by isomerization to the(E,E)-diene. Alternatively, compound 7 can be coupled with 20 viaPd-coupling to form phosphonate 19 which can be used in a Wittig-typereaction with aldehyde 8 followed by isomerization to form 10.

The final assembly of trihydroxy polyunsaturated eicosanoids and theiranalogs can be done as shown in Scheme 11. Sonogashira-type coupling ofthe two key intermediates 3 and 4, followed by deprotection gives thebis-alkynyl product of type 2. The final compound of type 1 can beobtained via selective hydrogenation using hydrogen and Lindlar catalystor alternatively using activated zinc. The activated zinc is typicallyused in methanol or aqueous media and can be prepared from zinc,Cu(OAc)₂.H₂O and AgNO₃ using literature procedures (Boland, W. et al(1987) Helv. Chim. Acta 1987, 70, 1025; Alami, M. et al. (1997)Tetrahedron Asymmetry, 8, 2949; Rodriguez, A. R. et al (2001)Tetrahedron Lett., 42, 6057).

Another embodiment of the present invention involves the preparation ofisotopically labeled derivatives of lipid mediators, such as 1 and itsanalogs, by using activated zinc in the presence of isotopically labeledmedia, such as isotopically labeled water and isotopically labeledmethanol. For example, compound 2 can be converted to the tetra-deuterioderivative 1-D₄ by using activated zinc-D₂O-CD₃OD, while thecorresponding tritiated derivative 1-T₄ can be prepared similarly fromtritiated water. This labeling process, can also be used to prepareother isotopically labeled polyunsaturated lipid mediators, such aslipoxins, leukotrienes and other resolvin derivatives. Such isotopicallylabeled polyunsaturated lipid mediators are useful as spectrometric ormolecular probes for the detection and study of the biological actionsof these molecules. The present synthesis offers major experimentaladvantages over the prior art. In the general case, outlined below, thepresent method can be used to prepare a wide range of compounds of thegeneral formulas shown, wherein:

-   -   R^(a) is hydrogen, alkyl, aryl, heteroaryl, acyl, silyl,        alkoxyacyl and aminoacyl; and    -   R_(A) and R_(B) are independently selected from the group        consisting of alkyl, perfluoroalkyl, alkenyl, aryl or        heteroaryl.

Overall the provided synthetic methodology is highly convergent andallows a number of possible combinations of the key intermediates byusing Pd-mediated coupling processes.

The above methodology is highly versatile and it can be readily extendedto a variety of analogs of trihydroxy polyunsaturated eicosanoids thathave similar frameworks. Thus, in another aspect, the invention featuresmethods for the synthesis of compounds having the general formulas 23and 24, as outlined in Scheme 3. Compound 23 can be prepared via theselective hydrogenation of compound 24, which can be produced via aSonogashira-type coupling among compounds 25 and 26:

-   -   wherein:    -   R^(a), R^(b) and R^(c), are independently selected from the        group consisting of hydrogen, alkyl, aryl, heteroaryl, acyl,        silyl, alkoxyacyl and aminoacyl;    -   R¹, R² and R³ are independently selected from the group        consisting of hydrogen, alkyl, perfluoroalkyl, aryl and        heteroaryl;    -   Q is selected from the group consisting of:        -   —C(O)-A, —SO₂-A, —PO(OR)-A, where A is hydroxy, alkoxy,            aryloxy, amino, alkylamino, dialkylamino, or —OM, where M is            a cation selected from the group consisting of ammonium,            tetra-alkyl ammonium, Na, K, Mg, and Zn, and R is hydroxyl            or alkoxy;    -   Y, Z and W are linkers independently selected from the group        consisting of a ring containing up to 20 atoms and a chain of up        to 20 atoms, provided that Y, Z and W can independently include        one or more nitrogen, oxygen, sulfur or phosphorous atoms, and        further provided that Y, Z and W can independently include one        or more substituents selected from the group consisting of        hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro,        iodo, bromo, fluoro, hydroxy, alkoxy, aryloxy, carboxy, amino,        alkylamino, dialkylamino, acylamino, carboxamido, cyano, oxo,        thio, alkylthio, arylthio, acylthio, alkylsulfonate,        arylsulfonate, phosphoryl, and sulfonyl, and further provided        that Y, Z and W can also contain one or more fused carbocyclic,        heterocyclic, aryl or heteroaryl rings, and provided that all        linkers Y are connected to the adjacent C(R)OR group via a        carbon atom;    -   X is Cl, Br or I; and    -   G is selected from the group consisting of hydrogen, alkyl,        perfluoroalkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro,        iodo, bromo, fluoro, hydroxy, alkoxy, aryloxy, carboxy, amino,        alkylamino, dialkylamino, acylamino, and carboxamido.

In some embodiments, the invention provides a method for the synthesisof compounds of general formulas 27 and 28 (Scheme 12), wherein:

-   -   A is hydroxy, alkoxy, aryloxy, amino, alkylamino, dialkylamino,        or —OM, where M is a cation selected from the group consisting        of ammonium, tetra-alkyl ammonium, Na, K, Mg, and Zn; and    -   R^(a), R^(b) and R^(c), are independently selected from the        group consisting of hydrogen, alkyl, aryl, heteroaryl, acyl,        silyl, alkoxyacyl and aminoacyl; and

As outlined in Scheme 12, compound 27 can be prepared via the selectivehydrogenation of compound 28, which can be performed by treatingcompound 28 with hydrogen and Lindlar catalyst or by using activatedzinc in the presence of an alcohol such as methanol, or using an aqueousmedium. The activated zinc reagent suitable for this process can beprepared from zinc, a copper salt, such as copper acetate and a silversalt, such as silver nitrate.

Compounds of the general formula 28 can be prepared via aSonogashira-type coupling among a compound of formula 29 and a compoundof formula 4, where X is Cl, Br or I. For example compounds 29 and 4 canbe converted to 28, upon treatment with a palladium catalyst, such astetrakis(triphenyl phosphine)palladium, in the presence of a copper saltsuch as copper(I) iodide, and an amine base such as triethylamine.

The invention also provides methods for the preparation of compound offormula 29 or its analogs. Compound 29 can be prepared via severalmethods which are outlined in Scheme 12. One such method involves theWittig-type coupling among an aldehyde compound of formula 30 and aphosphonate compound of formula 31, followed by desilylation. Compound31 can be formed via the Sonogashira-type coupling among compound 32 andalkyne compound 33.

In another embodiment, compound 29 can be prepared via the directSonogashira-type coupling among alkyne compound 33 and compound offormula 34. Alternatively, compound 33 can be coupled to compound 37 toform compound 36 which can undergo a Suzuki-type coupling with compound35 to produce, after desilylation, the key compound 29. Compound offormula 34 can be prepared by several methods, including the Wittig-typecoupling between aldehyde 30 and phosphonate 31, and thepalladium-mediated homologation of compound 35.

Compound 29 can also be prepared via the addition of an allenylorganoboron derivative or other allenyl organometallic derivative 11 toaldehyde 38, which can be prepared form 33.

In compounds shown in Scheme 12,

-   -   A is hydroxy, alkoxy, aryloxy, amino, alkylamino, dialkylamino,        or —OM, where M is a cation selected from the group consisting        of ammonium, tetra-alkyl ammonium, Na, K, Mg, and Zn;    -   R^(a) and R^(b) are independently selected from the group        consisting of hydrogen, alkyl, aryl, heteroaryl, acyl, silyl,        alkoxyacyl and aminoacyl; and    -   X is Cl, Br or I;    -   R^(d) is alkyl or aryl; and    -   R^(e) is hydrogen, alkyl or aryl; and    -   each of the three R groups in SiR₃ is independently selected        from a group consisting of alkyl, aryl and alkoxy; and    -   M² is magnesium zinc, copper, tin, silicon or boron.

Trihydroxy Polyunsaturated Eicosanoid Analogs

The invention also provides compounds and compositions containingsynthetic analogs of trihydroxy polyunsaturated eicosanoids that aresynthetic derivatives or analogs of compound 1 and exhibit improvedchemical and biological properties. The provided compounds includederivatives having the general formulas 27 and 28.

wherein:

-   -   A is hydroxy, alkoxy, aryloxy, amino, alkylamino, dialkylamino,        or —OM, where M is a cation selected from the group consisting        of ammonium, tetra-alkyl ammonium, Na, K, Mg, and Zn; and    -   R^(a), R^(b) and R^(c), are independently selected from the        group consisting of hydrogen, alkyl, aryl, heteroaryl, acyl,        silyl, alkoxyacyl and aminoacyl.

The invention also provides compounds having the general formulas 23 and24, as well as methods for their preparation and use.

wherein,

-   -   A is hydroxy, alkoxy, aryloxy, amino, alkylamino, dialkylamino,        or —OM, where M is a cation selected from a group consisting of        ammonium, tetra-alkyl ammonium, Na, K, Mg, or Zn;    -   Ra, Rb and Rc, are independently selected from a group that        consists of hydrogen, alkyl, aryl, heteroaryl, acyl, silyl,        alkoxyacyl or aminoacyl;    -   R¹, R² and R³ are independently selected from a group that        consists of hydrogen, alkyl, perfluoroalkyl, aryl or heteroaryl;    -   Q is selected from a group that consists of:        -   —C(O)-A, —SO2-A, —PO(OR)-A, where A is hydroxy, alkoxy,            aryloxy, amino, alkylamino, dialkylamino, or —OM, where M is            a cation selected from a group consisting of ammonium,            tetra-alkyl ammonium, Na, K, Mg, or Zn; and R is hydroxyl or            alkoxy;    -   Y, Z and W are linkers selected from a group consisting of a        ring or a chain of up to 20 atoms that may include one or more        nitrogen, oxygen, sulfur or phosphorous atoms, provided that        linker A can have one or more substituents selected from the        group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,        heteroaryl, chloro, iodo, bromo, fluoro, hydroxy, alkoxy,        aryloxy, carboxy, amino, alkylamino, dialkylamino, acylamino,        carboxamido, cyano, oxo, thio, alkylthio, arylthio, acylthio,        alkylsulfonate, arylsulfonate, phosphoryl, and sulfonyl, and        further provided that the linker may also contain one or more        fused rings, including carbocyclic, heterocyclic, aryl or        heteroaryl rings, provided that all linkers Y are connected to        the adjacent C(R)OR group via a carbon atom;    -   G is selected from a group that consists of hydrogen, alkyl,        perfluoroalkyl, alkenyl, alkynyl, aryl, heteroaryl, chloro,        iodo, bromo, fluoro, hydroxy, alkoxy, aryloxy, carboxy, amino,        alkylamino, dialkylamino, acylamino, and carboxamido.

In certain embodiments, Y, Z and W are each alkylene which can besubstituted or unsubstituted. In other embodiments, Y, Z and W areselected from methylene, ethylene and propylene. In other embodiments, Yis methylene. In other embodiments, Z is propylene. In otherembodiments, W is ethylene.

Some preferred embodiments of the present invention provide compoundshaving the general formulas 39 and 40, as well as methods for theirpreparation and use.

-   -   wherein:    -   A is hydroxy, alkoxy, aryloxy, amino, alkylamino, dialkylamino,        or —OM, where M is a cation selected from the group consisting        of ammonium, tetra-alkyl ammonium, Na, K, Mg, and Zn; and    -   R^(a), R^(b) and R^(c), are independently selected from the        group consisting of hydrogen, alkyl, aryl, heteroaryl, acyl,        silyl, alkoxyacyl and aminoacyl;    -   R³ is selected from the group consisting of hydrogen, alkyl,        perfluoroalkyl, aryl and heteroaryl;    -   R⁴ is selected from the group consisting of hydrogen, alkyl,        perfluoroalkyl, alkenyl, alkynyl, aryl, heteroaryl, fluoro,        hydroxy, alkoxy, aryloxy,    -   R⁵ is selected from the group consisting of (i)-(iv) as follows:        -   i) CH₂CH(R⁶)CH₂, where R⁶ is hydrogen, alkyl,            perfluoroalkyl, aryl, heteroaryl, fluoro, hydroxy or alkoxy;        -   ii) CH₂C(R⁶R⁷)CH₂, where R⁶ and R⁷ are each independently            alkyl, perfluoroalkyl, aryl, or fluoro, or R⁶ and R⁷ are            connected together to form a carbocyclic or heterocyclic            ring;        -   iii) CH₂OCH₂, CH₂C(O)CH₂, or CH₂CH₂; and        -   (iv) R⁵ is a carbocyclic, heterocyclic, aryl or heteroaryl            ring; and

In certain embodiments, R⁴ is selected from the group consisting ofhydrogen, methyl and trifluoromethyl.

Other preferred embodiments of the present invention provide compoundshaving the general formulas 41 and 42, as well as methods for theirpreparation and use.

-   -   wherein:    -   A is hydroxy, alkoxy, aryloxy, amino, alkylamino, dialkylamino,        or —OM, where M is a cation selected from the group consisting        of ammonium, tetra-alkyl ammonium, Na, K, Mg, and Zn; and    -   R^(a), R^(b) and R^(c), are independently selected from the        group consisting of hydrogen, alkyl, aryl, heteroaryl, acyl,        silyl, alkoxyacyl and aminoacyl;    -   R² and R³ are independently selected from the group consisting        of hydrogen, alkyl, perfluoroalkyl, aryl and heteroaryl;    -   R⁴ is selected from the group consisting of hydrogen, alkyl,        perfluoroalkyl, alkenyl, alkynyl, aryl, heteroaryl, fluoro,        hydroxy, alkoxy, aryloxy,    -   R⁵ is selected from the group consisting of (i)-(iv) as follows:        -   i) CH₂CH(R⁶)CH₂, where R⁶ is hydrogen, alkyl,            perfluoroalkyl, aryl, heteroaryl, fluoro, hydroxy or alkoxy;        -   ii) CH₂C(R⁶R⁷)CH₂, where R⁶ and R⁷ are each independently            alkyl, perfluoroalkyl, aryl, or fluoro, or R⁶ and R⁷ are            connected together to form a carbocyclic or heterocyclic            ring;        -   iii) CH₂OCH₂, CH₂C(O)CH₂, or CH₂CH₂; and        -   (iv) R⁵ is a carbocyclic, heterocyclic, aryl or heteroaryl            ring; and    -   R⁸ and R⁹ are independently selected from the group consisting        of hydrogen, alkyl, perfluoroalkyl, alkoxy, aryl and heteroaryl,        or R⁸ and R⁹ are connected together to form a carbocyclic or        heterocyclic ring.

In certain embodiments, R⁸ and R⁹ are each independently selected fromthe group consisting of hydrogen, methyl and trifluoromethyl.

In some embodiments the present invention provides compounds of generalformulas 29 or 43-47:

wherein:

-   -   A is hydroxy, alkoxy, aryloxy, amino, alkylamino, dialkylamino,        or —OM, where M is a cation selected from the group consisting        of ammonium, tetra-alkyl ammonium, Na, K, Mg, and Zn; and    -   R^(a) and R^(b) are independently selected from the group        consisting of hydrogen, alkyl, aryl, heteroaryl, acyl, silyl,        alkoxyacyl and aminoacyl;    -   R² is hydrogen, alkyl, perfluoroalkyl, aryl and heteroaryl;    -   R⁵ is selected from the group consisting of (i)-(iv) as follows:        -   i) CH₂CH(R⁶)CH₂, where R⁶ is hydrogen, alkyl,            perfluoroalkyl, aryl, heteroaryl, fluoro, hydroxy or alkoxy;        -   ii) CH₂C(R⁶R⁷)CH₂, where R⁶ and R⁷ are each independently            alkyl, perfluoroalkyl, aryl, or fluoro, or R⁶ and R⁷ are            connected together to form a carbocyclic or heterocyclic            ring;        -   iii) CH₂OCH₂, CH₂C(O)CH₂, or CH₂CH₂; and        -   (iv) R⁵ is a carbocyclic, heterocyclic, aryl or heteroaryl            ring; and    -   R⁸ and R⁹ are independently selected from the group consisting        of hydrogen, alkyl, perfluoroalkyl, alkoxy, aryl and heteroaryl,        or R⁸ and R⁹ are connected together to form a carbocyclic or        heterocyclic ring; and    -   each of the three R groups in SiR₃ is independently selected        from a group consisting of alkyl, aryl and alkoxy.

Pharmaceutical Compositions

The compounds of the invention can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the active compound and a pharmaceutically acceptable carrier.As used herein the language “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the treatment methods of the invention, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Therapeutic Uses

The compounds of the invention are derivatives or structural analogs ofnaturally-occurring trihydroxy polyunsaturated eicosanoids that areknown to have biological activity against a wide variety of targets,including diseases or conditions associated with inflammation orinflammatory response, autoimmune diseases, rheumatoid arthritis,cardiovascular diseases, or abnormal cell proliferation or cancer. Assuch, the compounds of the invention are expected to have similaractivity against those targets.

Accordingly, in one aspect the invention features methods ofameliorating or treating diseases or conditions associated withinflammation or inflammatory response, involving the administration to asubject of a therapeutically effective amount of a compound or compoundsof the invention, such that inflammation or an inflammatory response aresignificantly reduced or eliminated in the subject. A significantreduction includes the reduction or elimination of a symptom or symptomsassociated with the inflammation or inflammatory response.

In another aspect, the invention features methods of ameliorating ortreating diseases or conditions associated with abnormal cellproliferation, such as cancer, involving the administration to a subjectof an effective amount of a compound or compounds of the invention. Ingeneral, an effective amount is an amount sufficient to ensure adequateexposure of a target cell population, such that abnormal cellproliferation is substantially slowed or halted. A target population isa population of cells undergoing abnormal cell proliferation, such ascancerous and/or tumorous growth.

The invention will be further described in the following examples, whichare illustrative only, and which are not intended to limit the scope ofthe invention described in the claims.

EXAMPLES

The invention will be further described in the following examples, whichare illustrative only, and which are not intended to limit the scope ofthe invention described in the claims.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees centigrade,and pressure is at or near atmospheric. Starting materials used in theseexamples are generally either commercially available or can be readilyprepared from commercially available reagents by a procedure involvingone or more steps.

Example 1 (5S,8E,10E,12R/S)-methyl5,12-bis(tert-butyldimethylsilyloxy)pentadeca-8,10-dien-6,14-diynoate

Step 1: To a solution of (2E,4E)-5-bromopenta-2,4-dien-1-ol (0.74 g,4.51 mmol) in Et₂NH (8 ml) was added Pd(PPh₃)₄ (160 mg, 0.14 mmol) andthe solution protected from light was stirred for 45 minutes at roomtemperature. A small amount of benzene (4 ml) was added to completelydissolve the catalyst. To the resulting homogeneous solution was thenadded through a cannula a solution of (S)-methyl5-(tert-butyldimethylsilyloxy)hept-6-ynoate (1.25 g, 4.61 mmol) in Et₂NH(8 ml) and CuI (88 mg, 0.46 mmol). The mixture was stirred for 3 h atroom temperature and quenched with a saturated aqueous solution ofammonium chloride and extracted with ether. It was then washed withbrine, dried and concentrated. Flash column chromatography (silica gel,20% ethyl acetate/hexanes) afforded the pure product as a colorlessliquid (1.52 g, 4.33 mmol, 96% yield). ¹H NMR (360 MHz, CDCl₃): δ 6.61(dd, J=15.7 Hz and 10.6 Hz, 1H), 6.02 (dd, J=14.8 Hz and 10.9 Hz, 1H),5.57 (d, J=14.4 Hz), 5.48 (dt, J=15.1 Hz and 5.2 Hz, 1H), 4.54 (m, 1H),3.75 (s, 2H), 3.30 (s, 3H), 2.14 (t, J=7.0 Hz, 2H), 1.85 (m, 2H), 1.77(m, 2H), 1.05 (s, 9H), 0.27 (s, 3H), 0.18 (s, 3H). ¹³C NMR (62 MHz,CDCl₃): δ 141.705, 136.071, 129.466, 111.138, 93.991, 84.393, 63.859,62.789, 51.161, 38.516, 33.831, 26.244, 21.391, −3.936, −4.648.

Step 2: To a solution of dimethyl sulfoxide (0.66 ml, 8.5 mmol) inCH₂Cl₂ (40 ml) was added dropwise at −78° C. oxalyl chloride (0.56 ml,6.4 mmol) and the reaction was stirred at that temperature for 15minutes. Alcohol from Step 1 (1.5 g, 4.26 mmol) was added via adouble-tipped needle and the resulting solution was stirred anadditional 45 minutes at −78° C. Triethylamine (2.96 ml, 21.3 mmol) wasadded slowly to the cloudy white mixture that was allowed to warm up toroom temperature and it was then poured into water and extracted withethyl acetate. The combined extracts were dried and concentrated. Flashcolumn chromatography (silica gel, 5% ethyl acetate/hexanes) affordedthe pure product as a colorless liquid (1.31 g, 3.75 mmol, 87% yield).¹H NMR (360 MHz, C₆D₆): δ 9.50 (d, J=8.2 Hz, 1H), 6.31 (dd, J=14.7 Hzand 11.3 Hz, 1H), 6.22 (dd, J=14.6 Hz and 11.2 Hz, 1H), 5.77 (dd, J=14.9Hz and 8.2 Hz, 1H), 5.59 (d, J=15.9 Hz, 1H), 4.50 (m, 1H), 3.34 (s, 3H),2.16 (t, J=7.0 Hz, 2H), 1.85 (m, 2H), 1.76 (m, 2H), 1.04 (s, 9H), 0.26(s, 3H), 0.18 (s, 3H). ¹³C NMR (62 MHz, CDCl₃): δ 191.944, 172.975,148.566, 138.933, 133.120, 119.950, 98.691, 83.368, 63.524, 50.987,38.019, 33.504, 25.921, 21.021, 18.370, −4.320, −4.931.

Step 3: To a solution of the allenyl boronic acid (518 mg, 6.18 mmol) intoluene (20 ml) were added molecular sieves (3.0 g) anddiisopropyl-D-tartrate (2.6 ml, 12.36 mmol) and the resulting solutionwas allowed to stand at room temperature for 24 h with gentle stirringfrom time to time. The obtained solution of chiral allenyl boronic esterwas then cannulated to a new flask and cooled at −78° C. At this point asolution of the aldehyde from Step 2 (665 mg, 1.9 mmol) in toluene (10ml) was added through a double tipped needle and the reaction mixturewas stirred at −78° C. for 12 h and then warmed up slowly at roomtemperature overnight. The resulting solution was then quenched with adiluted solution of HCl, extracted with ether and it was then washedwith brine, dried and concentrated. Flash column chromatography (silicagel, 5% ethyl acetate/hexanes) afforded the pure product as a colorlessliquid (592 mg, 1.52 mmol, 80% yield). To a solution of the obtainedalcohol product (592 mg, 1.52 mmol) in CH₂Cl₂ (10 ml) were addeddropwise at 0° C. 2,6-lutidine (0.40 ml, 3.34 mmol) andtert-butyldimethyl-silyloxy triflate (0.41 ml, 2.28 mmol). The reactionmixture was warmed up to room temperature and stirred for 4 hours. Theresulting solution was then poured into a solution of saturated NH₄Cland extracted with diethyl ether. The combined extracts were dried andconcentrated. Flash column chromatography (silica gel, 2% ethylacetate/hexanes) afforded the product as a colorless liquid in 95%yield. ¹H NMR (250 MHz, CDCl₃): δ 6.52 (dd, J=15.5 Hz and 10.9 Hz, 1H),6.26 (dd, J=15.2 and 1.0 Hz, 1H), 5.85 (dd, J=15.5 Hz and 5.3 Hz, 1H),5.10 (d, J=16.2 Hz, 1H), 4.51 (t, J=5.6 Hz, 1H), 4.31 (q, J=5.9 Hz, 1H),3.54 (s, 3H), 2.45 (m, 4H), 1.95 (t, J=1.4 Hz, 1H), 1.82 (m, 4H), 0.97(s, 18H), 0.18 (s, 3H), 0.12 (s, 3H), 0.07 (s, 3H), 0.05 (s, 3H). ¹³CNMR (62 MHz, CDCl₃): δ 173.891, 140.738, 137.433, 129.200, 111.125,93.363, 83.432, 80.947, 71.306, 70.197, 63.012, 51.452, 37.889, 33.516,28.296, 25.792, 20.566, 18.075, −4.419, −4.578, −4.861, −5.014.

Example 2 (5S,8E,10E,12R)-methyl5,12-bis(tert-butyldimethylsilyloxy)pentadeca-8,10-dien-6,14-diynoate

Step 1: To a solution of(R,1E,3E)-1-bromo-5-(tert-butyldimethylsilyloxy)-8-(trimethylsilyl)octa-1,3-dien-7-yne(100 mg, 0.26 mmol) in benzene (1 ml) was added Pd(PPh₃)₄ (15 mg, 0.013mmol) and the reaction protected from light was stirred for 45 minutesat room temperature. To the resulting solution was then added through acannula a solution of (S)-methyl5-(tert-butyldimethylsilyloxy)hept-6-ynoate (105 mg, 0.39 mmol) inbenzene (1 ml), CuI (12 mg, 0.063 mmol) and triethylamine (0.4 g, 4mmol). The mixture was stirred for 3 hr at room temperature and quenchedwith a saturated aqueous solution of ammonium chloride and extractedwith ether. It was then washed with brine, dried and concentrated. Flashcolumn chromatography (silica gel, 4% diethyl ether/hexanes) affordedthe pure product as a colorless liquid (127 mg, 0.22 mmol, 85% yield).

Step 2: To a solution of the product from Step 1 (127 mg, 0.22 mmol) inTHF/EtOH (2 ml/1 ml) was added a solution of silver nitrate (106 mg,0.63 mmol) in water/EtOH (1 ml/1 ml) at 0° C. The resulting yellow solidsuspension was allowed to warm to 25° C. and it was then treated with asolution of potassium cyanide (71 mg, 1.09 mmol) in water (1 ml). Theproduct was extracted with ether, washed with brine, dried andconcentrated. Flash column chromatography (silica gel, 4% diethylether/hexanes) afforded the pure product as a colorless liquid in 89%yield. ¹H NMR (250 MHz, C₆D₆): δ 6.58 (dd, J=15.3 Hz and 10.9 Hz, 1H),6.14 (dd, J=16.0 and 11.0 Hz, 1H), 5.65 (dd, J=16.3 Hz and 6.3 Hz, 1H),5.56 (d, J=16.0 Hz, 1H), 4.52 (t, J=7.5 Hz, 1H), 4.20 (q, J=6.4 Hz, 1H),3.34 (s, 3H), 2.20 (m, 4H), 2.12 (t, J=1.4 Hz, 1H), 1.78 (m, 4H), 1.03(s, 9H), 0.97 (s, 9H), 0.25 (s, 3H), 0.17 (s, 3H), 0.06 (s, 3H), 0.02(s, 3H); ¹³C NMR (62 MHz, CDCl₃): δ 173.891, 140.738, 137.433, 129.200,111.125, 93.363, 83.432, 80.947, 71.306, 70.197, 63.012, 51.452, 37.889,33.516, 28.296, 25.792, 20.566, 18.075, −4.419, −4.578, −4.861, −5.014.

Example 3 (5S,8E,10E,12R)-methyl5,12-bis(tert-butyldimethylsilyloxy)pentadeca-8,10-dien-6,14-diynoate

Step 1: A mixture of 3-bromo-propene bromide (0.5 g, 2.5 mmol) andtriethylphosphite (neat, 0.83 g, 5 mmol) was heated to 120° C. for 3 hr.The excess phosphate was removed under vacuum and used directly in nextstep.

Step 2: To a solution of the phosphonate product of Step 1 (257 mg, 1.0mmol) in 7 ml dry benzene, was added (S)-methyl5-(tert-butyldimethylsilyloxy)hept-6-ynoate (270 mg, 11.0 mmol),tetrakis(triphenyl phosphine)palladium, (230 mg, 0.2 mmol), copper(I)iodide, (76 mg, 0.4 mmol), and triethylamine (1.01 g, 10 mmol). Themixture was stirred at room temperature, overnight. Removal of thesolvent and column chromatography (1% MeOH in diethyl ether) gave thecoupled phosphonate product (220 mg, 60%). This compound exhibitedsatisfactory spectroscopic and analytical data.

Step 3: To a solution of phosphonate from Step 2 (217 mg 0.486 mmol) in3 ml dry THF, cooled to −78° C. was added 0.51 ml 1M sodiumbis(trimethylsily)amide (0.55 mmol). The reaction mixture was stirredfor 3 min and freshly prepared(R)-2-(tert-butyldimethylsilyloxy)-5-(trimethylsilyl)pent-4-ynal (136mg, 0.5 mmol) in 2.5 ml THF was added The mixture was stirred at −78° C.for 3 hrs, warmed up to room temperature, and stirred for another 30mins. Sat. NH4Cl aqueous solution was added, and the mixture wasextracted with ether. Removal of the solvent under vacuum and columnchromatography (3% ethyl acetate in hexanes) gave 120 mg (43%) of theproduct.

Step 4: The product of Step 3 was treated similarly to Example 2, Step 2to give (5S,8E,10E,12R)-methyl5,12-bis(tert-butyldimethylsilyloxy)pentadeca-8,10-dien-6,14-diynoate.

Example 4 (5S,8E,10E,12R,16E,18R)-methyl5,12,18-tris(tert-butyldimethylsilyloxy)icosa-8,10,16-trien-6,14-diynoate

Sonogashira coupling between(R,E)-tert-butyl(1-iodopent-1-en-3-yloxy)dimethylsilane and(5S,8E,10E,12R)-methyl5,12-bis(tert-butyldimethylsilyloxy)pentadeca-8,10-dien-6,14-diynoate(the product of Example 2 or Example 3), using a procedure analogous tothat of Example 2, Step 1 gave the product in 80% yield. ¹H NMR (500MHz, C₆D₆): δ 6.59 (dd, J=15.2 Hz and 10.9 Hz, 1H), 6.24 (dd, J=15.2 and11.0 Hz, 1H), 6.14 (dd, J=15.5 Hz and 5.3 Hz, 1H), 5.86 (d, J=15.4 Hz,1H), 5.67 (dd, J=14.8 Hz and 5.6 Hz, 1H), 5.59 (d, J=15.5 Hz, 1H), 4.54(t, J=5.7 Hz, 1H), 4.24 (q, J=5.9 Hz, 1H), 3.94 (q, J=5.6 Hz, 1H), 3.35(s, 3H), 2.46 (m, 2H), 2.17 (t, J=7.1 Hz, 2H), 1.84 (m, 4H), 1.44 (m,2H), 1.04 (s, 9H), 1.02 (s, 9H), 1.00 (s, 9H), 0.86 (t, J=7.5 Hz, 3H),0.28 (s, 3H), 0.19 (s, 3H), 0.14 (s, 3H), 0.08 (s, 3H), 0.07 (s, 3H),0.05 (s, 3H). ¹³C NMR (125 MHz, C₆D₆): δ 173.131, 145.665, 141.248,138.411, 129.420, 111.518, 109.904, 93.989, 87.526, 84.119, 81.048,73.998, 72.025, 63.570, 50.913, 38.321, 33.587, 31.253, 29.235, 26.014,21.163, 18.413, 9.221, −4.207, −4.421, −4.603, −4.621, −4.772, −5.094.

Example 5 (5S,8E,10E,12R,16E,18R)-methyl5,12,18-trihydroxyicosa-8,10,16-trien-6,14-diynoate

A solution of the product of Example 4 (40 mg, 0.065 mmol) in THF (1 ml)was treated with 1.0 M TBAF (0.32 ml, 0.32 mmol) at 0° C. The reactionwas stirred for 3 h and then poured into water and extracted with ether.The ether extracts were washed with brine, dried and concentrated. Theethereal solution was then treated with an excess of freshly prepareddiazomethane in ether to convert the free acid to the product. Flashcolumn chromatography (silica gel, 4% MeOH/CH₂Cl₂) afforded the pureproduct in 90% yield. ¹H NMR (500 MHz, C₆D₆): δ 6.55 (dd, J=15.5 Hz and10.9 Hz, 1H), 6.16 (dd, J=15.2 Hz and 11.0 Hz, 1H), 6.05 (dd, J=15.5 Hzand 5.3 Hz, 1H), 5.70 (d, J=16.2 Hz, 1H), 5.61 (dd, J=14.6 Hz and 5.5Hz, 1H), 5.58 (d, J=14.7 Hz, 1H), 4.28 (t, J=5.8 Hz, 1H), 4.06 (dd,J=11.2 Hz and 5.3 Hz, 1H), 3.65 (dd, J=11.0 Hz and 6.7 Hz, 1H), 3.30 (s,3H), 2.36 (m, 2H), 2.06 (t, J=6.9 Hz, 2H), 1.72 (m, 2H), 1.59 (m, 2H),1.27 (m, 2H), 0.74 (t, J=7.4 Hz, 3H). ¹³C NMR (125 MHz, C₆D₆): δ173.819, 145.219, 141.143, 136.647, 130.007, 111.340, 109.915, 92.672,85.857, 84.082, 81.330, 73.505, 70.225, 62.533, 51.488, 37.097, 33.599,29.912, 28.658, 20.615, 9.451. HPLC: Beckman Ultrasphere reverse phasecolumn (30% water in MeOH, 3.8 ml/min, 252 bar): elution time=5.41 min.

Example 6 (5S,6Z,8E,10E,12R,14Z,16E,18R)-methyl5,12,18-trihydroxyicosa-6,8,10,14,16-pentaenoate or resolvin E1 methylester

To a solution of the bis-acetylenic product from Example 5 (7.7 mg,0.021 mmol) in dichloromethane (4 ml) was added Lindlar catalyst (1.5mg, 20% by weight), quinoline (4 μl), and the reaction mixture wasstirred under the static atmosphere of hydrogen. Samples were takenevery 20 minutes for HPLC analysis (30% water in MeOH), and the reactionwas stopped at 60% conversion. The resulting solution was filtrated overa pad of celite and separated by HPLC (45% water in MeOH) affording thepure product in 60% yield. ¹H NMR (500 MHz, C₆D₆): δ 6.54 (dd, J=14.8 Hzand 11.5 Hz, 1H), 6.49 (dd, J=14.9 Hz and 11.7 Hz, 1H), 6.26 (dd, J=16.0Hz and 10.5 Hz, 1H), 6.11 (t, J=9.2 Hz, 1H), 6.09 (dd, J=14.7 Hz and11.1 Hz, 1H), 5.95 (t, J=11.0 Hz, 1H), 5.60 (dd, J=15.4 Hz and 6.4 Hz,1H), 5.56 (dd, J=14.9 Hz and 6.0 Hz, 1H), 5.42 (dt, J=10.8 Hz and 8.1Hz, 1H), 5.30 (t, J=10.6 Hz, 1H), 4.38 (q, J=7.8 Hz, 1H), 4.03 (q, J=6.6Hz, 1H), 3.83 (q, J=6.6 Hz, 1H), 3.30 (s, 3H), 2.2-2.4 (m, 4H), 2.08 (t,J=6.9 Hz, 2H), 1.6-1.7 (m, 2H), 1.3-1.5 (m, 2H), 0.85 (t, J=6.7 Hz, 3H).¹³C NMR (125 MHz, C₆D₆): δ 177.135, 137.855, 137.106, 134.923, 134.057,131.093, 130.273, 129.637, 128.428, 126.868, 125.269, 73.554, 71.747,67.609, 37.123, 36.223, 33.835, 30.576, 21.165, 9.867. HPLC: BeckmanUltrasphere reverse phase column (30% water in MeOH, 3.8 ml/min, 254bar): elution time=8.43 min.

Example 7 (5S,6Z,8E,10E,12R,14Z,16E,18R)-Methyl6,7,14,15-tetradeuterio-5,12,18-trihydroxyicosa-6,8,10,14,16-pentaenoate

Step 1: To zinc dust (<10 micron, 98+%, Aldrich, 20, 998-8) that wasweighed under nitrogen, is added deuterated water ((D₂O, 3 mL), whichwas previously degassed with bubbling nitrogen for 20 minutes. Afterstirring for 15 min, copper(II) acetate monohydrate (Ac₂Cu.H₂O, 98+%,Aldrich, 6046-93-1, 50 mg) was added and the mixture was stirred foranother 20 min. To the stirred mixture is added slowly silver (I)nitrate, (AgNO₃, 99+%, Aldrich, 20,913-9, 50 mg). The mixture wasstirred for another 30 min and the precipitate was collected byfiltration and rinsed with deuterated water (2×3 mL), acetone (2×3 mL),and ether (2×3 mL). The precipitate was mixed with 2.5 mL of a 4:1mixture by volume of deuterated water:dioxane (1,4-dioxane, anhydrous,99.8+%, Aldrich, 296309-1L).

Step 2: The Zn—Cu—Ag reagent prepared according to Step 1 was added to asolution of (5S,8E,10E,12R,16E,18R)-methyl5,12,18-trihydroxyicosa-8,10,16-trien-6,14-diynoate from Example 5, (0.5mg) in dioxane (0.5 mL) and the mixture was stirred at 40° C. for 24 hr.The mixture is then filtered through a glass fritted funnel, thefiltrate is evaporated and the crude product is purified by HPLC to give(5S,6Z,8E,10E,12R,14Z,16E,18R)-Methyl6,7,14,15-tetradeuterio-5,12,18-trihydroxyicosa-6,8,10,14,16-pentaenoate.This compound gave satisfactory spectra.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A method of ameliorating or treating a disease or conditionassociated with inflammation, inflammatory response, autoimmune disease,rheumatoid arthritis, cardiovascular disease, abnormal cellproliferation, or cancer, the method comprising administering to asubject an effective amount of a compound having a structure of generalformula 46:

wherein: A is hydroxy, alkoxy, aryloxy, amino, alkylamino, dialkylamino,or —OM, where M is a cation selected from ammonium, tetra-alkylammonium, Na, K, Mg, and Zn; R^(a) and R^(b) are independently selectedfrom hydrogen, alkyl, aryl, heteroaryl, acyl, silyl, alkoxyacyl andaminoacyl; R² is hydrogen, alkyl, perfluoroalkyl, aryl or heteroaryl; R⁵is selected from i)-iv) as follows: i) CH₂CH(R⁶)CH₂, where R⁶ ishydrogen, alkyl, perfluoroalkyl, aryl, heteroaryl, fluoro, hydroxy oralkoxy; ii) CH₂C(R⁶R⁷)CH₂, where R⁶ and R⁷ are each independently alkyl,perfluoroalkyl, aryl, or fluoro, or R⁶ and R⁷ are connected together toform a carbocyclic or heterocyclic ring; iii) CH₂OCH₂, CH₂C(O)CH₂, orCH₂CH₂; and iv) a carbocyclic, heterocyclic, aryl or heteroaryl ring;and R⁸ and R⁹ are independently selected from hydrogen, alkyl,perfluoroalkyl, alkoxy, aryl and heteroaryl, or R⁸ and R⁹ are connectedtogether to form a carbocyclic or heterocyclic ring.
 2. The method ofclaim 1, wherein R^(a) and R^(b) are independently selected fromhydrogen, acyl, alkoxyacyl and aminoacyl.
 3. The method of claim 1,wherein the compound has a structure of general formula 44,


4. The method of claim 3, wherein R^(a) and R^(b) are independentlyselected from hydrogen, acyl, alkoxyacyl and aminoacyl.
 5. The method ofclaim 3 for ameliorating or treating a disease or condition associatedwith inflammation.
 6. The method of claim 3, wherein the compound has astructure of general formula 29,


7. The method of claim 6, wherein R^(a) and R^(b) are independentlyselected from hydrogen, acyl, alkoxyacyl and aminoacyl.
 8. The method ofclaim 6 for ameliorating or treating a disease or condition associatedwith inflammation.
 9. A method of ameliorating or treating a disease orcondition associated with inflammation, inflammatory response,autoimmune disease, rheumatoid arthritis, cardiovascular disease,abnormal cell proliferation, or cancer, the method comprisingadministering to a subject an effective amount of a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound having a structure of general formula 46:

wherein: A is hydroxy, alkoxy, aryloxy, amino, alkylamino, dialkylamino,or —OM, where M is a cation selected from ammonium, tetra-alkylammonium, Na, K, Mg, and Zn; R^(a) and R^(b) are independently selectedfrom hydrogen, alkyl, aryl, heteroaryl, acyl, silyl, alkoxyacyl andaminoacyl; R² is hydrogen, alkyl, perfluoroalkyl, aryl or heteroaryl; R⁵is selected from i)-iv) as follows: i) CH₂CH(R⁶)CH₂, where R⁶ ishydrogen, alkyl, perfluoroalkyl, aryl, heteroaryl, fluoro, hydroxy oralkoxy; ii) CH₂C(R⁶R⁷)CH₂, where R⁶ and R⁷ are each independently alkyl,perfluoroalkyl, aryl, or fluoro, or R⁶ and R⁷ are connected together toform a carbocyclic or heterocyclic ring; iii) CH₂OCH₂, CH₂C(O)CH₂, orCH₂CH₂; and iv) a carbocyclic, heterocyclic, aryl or heteroaryl ring;and R⁸ and R⁹ are independently selected from hydrogen, alkyl,perfluoroalkyl, alkoxy, aryl and heteroaryl, or R⁸ and R⁹ are connectedtogether to form a carbocyclic or heterocyclic ring.
 10. The method ofclaim 9, wherein R^(a) and R^(b) are independently selected fromhydrogen, acyl, alkoxyacyl and aminoacyl.
 11. The method of claim 9,wherein the compound has a structure of general formula 44,


12. The method of claim 11, wherein R^(a) and R^(b) are independentlyselected from hydrogen, acyl, alkoxyacyl and aminoacyl.
 13. The methodof claim 11 for ameliorating or treating a disease or conditionassociated with inflammation.
 14. The method of claim 11, wherein thecompound has a structure of general formula 29,


15. The method of claim 14, wherein R^(a) and R^(b) are independentlyselected from hydrogen, acyl, alkoxyacyl and aminoacyl.
 16. The methodof claim 14 for ameliorating or treating a disease or conditionassociated with inflammation.